FANCA Controls Mitotic Phosphosignaling Networks To Ensure Genome Stability During Cell Division

Abstract

Fanconi anemia (FA) is a heterogenous genome instability syndrome with a high risk of cancer. The FA proteins are essential for interphase DNA damage repair. However, it is incompletely understood why FA-deficient cells also develop gross aneuploidy and multinucleation, which are symptoms of error-prone chromosome segregation. Emerging evidence indicates that the FA signaling network functions as a guardian of the genome throughout the cell cycle, including chromosome segregation during mitosis. However, the mechanistic aspects of the critical role of the FA signaling in mitosis remain poorly understood.We have recently shown that the FA signaling network localizes to the mitotic apparatus to control the spindle assembly checkpoint and centrosome maintenance (J Clin Invest 2013, in press). The spindle assembly checkpoint (SAC) is a complex tumor suppressor signaling network that prevents premature separation of sister chromatids by delaying the metaphase-to-anaphase transition until all the kinetochores are properly attached to the mitotic spindle. Since weakened SAC promotes stochastic chromosome segregation, mutagenesis and cancer, these findings shed new light on the role of FA signaling in maintenance of genomic stability. We found the subcellular localization of FA proteins to the mitotic apparatus is spatiotemporally regulated as cells divide.Our new data revealed the pathways connecting the FANCA protein with canonical mitotic phosphosignaling networks. We have employed unbiased kinome-wide phospho-mass spectrometry to compare the landscape of abnormalities of mitotic signaling pathways in primary FANCA-/- patient cells and gene-corrected isogenic cells. These experiments led us to identify and quantify a wide range of phosphorylation abnormalities of multiple FANCA-dependent centrosome-, kinetochore- and chromosome-associated regulators of mitosis. Our data illuminated the role for FA signaling in three critical stages of cell division: (1) the spindle assembly checkpoint, (2) anaphase and (3) cytokinesis. Thus, we employed live phase-contrast imaging of primary FANCA-/- patient cells in comparison to gene-corrected cells to separately quantify aberrations in (1) chromosome congression and metaphase-anaphase transition (SAC malfunction), (2) execution of anaphase and (3) completion of cytokinesis.Our findings further our understanding of human cell cycle control and provide new insights into the origins of genomic instability in Fanconi anemia by establishing mechanistic connection between the FANCA protein and key mitotic signaling networks. The identification of cell division pathways regulated by FANCA has implications for future targeted drug development in Fanconi anemia and FA-deficient malignancies in the general population. Disclosures:No relevant conflicts of interest to declare.